I. Field of the Invention
The present invention relates to the general field of biochemical assays and separations, and to apparatus for their practice, generally classified in U.S. Patent Class 435.
II. Description of the Prior Art
Interest in nucleic acid purification and assays has increased with human trials of plasmid-based vaccines (e.g., for influenza, HIV, and malaria) and therapeutics (e.g., insulin and vascularization promoters) as well as the steady expansion of DNA sequencing activities, pharmacogenomics, and genetic testing. This invention embodies a rapid and efficient method of nucleic acid assay using selective precipitation by compaction agents.
Prior Art will include the following:    Murphy, J. C., Wibbenmeyer, J. A., Fox, G. E., and Willson, R. C. (1999) Purification of plasmid DNA using selective precipitation by compaction agents. Nature Biotechnol. 17, 822-823.    Murphy, J. C., G. E. Fox and R. C. Willson, “RNA Isolation and Fractionation using Compaction Agents,” Analytical Biochemistry, 295, 143 (2001).    Mourich D. V., Munks M. W., Murphy J. C., Willson R. C., and Hill A. B., “Spermine compaction is an efficient and economical method of producing vaccination-grade DNA”, J. Immunol. Methods. 274:257-264 (2003).    DeWalt, B., Murphy, J. C., Fox, G. E., and Willson, R. C., “Compaction Agent Clarification of Microbial Lysates”, Protein Expression and Purification, 28(2) 220-223 (2003).
Related U.S. Pat. No. 6,617,108 describes and claims processes for purification of DNA and separation of proteins, etc. by adding a compaction agent e.g. basic polypeptides, polyamines, trivalent and tetravalent metal ions to the mixture. This present application embodies the discovery that unexpectedly superior results are obtained by use of selected compaction agents e.g. quaternary ammonium compounds and the phosphonium-based catalysts. Preferred compaction agents include quaternary ammonium polyamines, the compounds of FIGS. 1-3. Tables B, C and D show some examples of these compounds.
In the literature, “q-value” and “C#” are useful parameters for correlating reactivity and selectivity. The q-value was proposed by Marc Halpern (PTC Organics) in 1988 and reflects the accessibility of the positive charge of a quat. A q-value of 1.0 to 2.0 often gives high reactivity for “transfer rate limited” reactions whereas a q-value of <1 often gives high reactivity for reactions which require a loose ion pair. The q-value is calculated by adding the reciprocals of the numbers of carbons on each of the 4 chains. C# is simply the sum of all of the carbons of the quat and is a measure of the organophilicity of the quat. Sources include Sachem, Cognis, Lion Akzo, Merck and PTC organics, whose websites give additional information.
III. Problems Presented by Prior Art
Most current methods of nucleic acid separation are relatively time-consuming and require the use of adsorbents, toxic substances, nucleases, and/or filtration media to separate individual nucleic acid types from protein, genomic DNA, endotoxins and especially the abundant RNA present in cell lysates. Many do not provide samples in a state ready for assays such as RT-PCR.
The present invention offers several important improvements over current methods: no DNAse and/or other enzymes need be used, the technique requires no chromatographic medium, and the purified samples are left in a form ready for assays such as RT-PCR. Also, with the use of different compaction agents, different types of nucleic acids can be separated from the same mixture. The invention can separate different types of RNA and DNA as long as some secondary structure is present.
In addition, RNA can be fractionated based on molecular weight via selective precipitation.
The method can also be used for parallel purification of a large number of samples (mini-preps) and is readily adaptable to automation (robotics).
In a preferred embodiment, the invention also provides a method for making a biochemical assay of one or more selected messenger RNA (mRNA) species, while reducing the RT-PCR background due to genomic DNA contamination.
The invention also provides a method of preparing a nucleic acid sample having reduced DNA content and also reduced content of compaction precipitation agent by selective removal of compaction agent.
Especially preferred embodiments include selective DNA precipitation over RNA, assay of RNA by first precipitating DNA, then assaying RNA in a second step; syntheses of preferred compaction precipitation agents; and removal of compaction agents before performing biochemical assays on the sample.